Methods and kits for improving global gene expression analysis of human urine derived rna

ABSTRACT

Disclosed are methods and kits for improving global gene expression analysis for a population of RNA molecules derived from a human urine sample. In an embodiment, the method comprises the step of selectively depleting miR-10a-5p fragments from the population of RNA molecules or selectively blocking miR-10a-5p fragments within the RNA population. The miR-10a-5p depleted or miR-10a-5p blocked population of RNA can be used in a variety of global gene expression analysis protocols, including next generation sequencing. In a further embodiment, the method comprises selectively depleting or blocking miR-10b-5p fragments within the RNA population. The miR-10a-5p and/or miR-10b-5p depleted or blocked populations of RNA can also be used in global gene expression analysis protocols, including next generation sequencing. The kit comprises oligonucleotide probes comprising a nucleotide sequence that is the complement to a nucleotide sequence of the miR-10a-5p and/or oligonucleotide probes comprising a nucleotide sequence that is the complement to a nucleotide sequence of miR-10b-5p.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a divisional of U.S. patent application Ser. No.16/042,282 filed Jul. 23, 2018, the subject matter of which isincorporated by reference herein in its entirety.

FIELD OF INVENTION

The present invention provides a method of improving global geneexpression analysis of human urine derived RNA, and in particular, thenext generation sequencing of small RNA.

BACKGROUND

Global expression profiling of RNA and small RNA from various bodilyfluids and tissue biopsies has become a staple approach for themonitoring and/or discovery of RNA biomarkers in various applications,including molecular diagnostics, dose/response effects studies, toxicitystudies and other related applications. Global gene expression analysiscan be carried out using a variety of methods including microarrayanalysis, library construction, reverse transcription, amplification,transcriptome profiling, expression analysis and sequencing, includingnext generation sequencing.

Human urine contains a variety of RNA molecules, which may be medicallyor scientifically relevant. The relative abundances of such RNAmolecules can be indicative of donor health status or responses tovarious endogenous and exogenous stimuli. Of the RNA molecules presentin human urine, a class of small non-coding, regulatory RNAs, calledmicroRNA (miRNA), are of particular interest as biomarkers. Interest inmiRNA as biomarkers is due to both their biological role in geneexpression regulation and their relative stability in circulation (ascompared to larger RNA molecules, which are more readily degraded).

Two miRNA molecules are abundant in the human urine miRNA milieu. Thesetwo molecules are: hsa-miR-10a and hsa-miR-10b (Zhou et al., 2017;El-Mogy et al., 2018). These molecules play a role in specific cancerssuch as colorectal cancer and renal cell carcinoma (Veerla et al., 2009;Xiao et al., 2014; Ma et al., 2015; Zhang et al., 2015;Abdelmaksoud-Dammak et al., 2017; Arai et al., 2017).

SUMMARY OF INVENTION

Disclosed are methods of improving global gene expression analysis for apopulation of RNA molecules derived from human urine. In one embodiment,the method comprises the step of depleting miR-10a-5p and/or miR-10b-5pfrom the population of RNA molecules. In another embodiment, the methodcomprises the step of blocking miR-10a-5p and/or miR-10b-5p fragments inthe population of RNA molecules. The method provides a sample, in whichthe miR-10a-5p and/or miR-10b-5p fragments are preferably blocked byhybridization with complementary oligonucleotide probes. The resultingmiR-10a-5p and/or miR-10b-5p depleted or blocked population of small RNAmolecules can be used in variety of downstream global gene expressionanalysis, and in particular, next generation sequencing.

In one aspect, disclosed is a method of improving global gene expressionanalysis for a population of RNA molecules derived from human urine, themethod comprising the step of depleting miR-10a-5p and/or miR-10b-5pfragments from the population of RNA molecules. The method may comprisedepleting only miR-10a-5p fragments from the population of RNAmolecules. The method may comprise depleting only miR-10b-5p fragmentsfrom the population of RNA molecules. The method may comprise depletingboth miR-10a-5p and miR-10b-5p fragments from the population of RNAmolecules.

In an embodiment of the method, the step of depleting miR-10a-5pfragments from the population of RNA molecules comprises:

-   -   adding miR-10a-5p specific oligonucleotide probes to a sample        containing the population of RNA molecules, wherein each        miR-10a-5p specific probe comprises a nucleotide sequence that        is the complement to a nucleotide sequence of miR-10a-5p;    -   forming a complex between one or more miR-10a-5p fragments and a        miR-10a-5p specific oligonucleotide probe; and    -   removing the miR-10a-5p:oligonucleotide complexes from the        sample.

Each miR-10a-5p specific oligonucleotide probe may comprise one ormultiple copies of a nucleotide sequence that is the complement to thenucleotide sequence of the miR-10a-5p.

In another embodiment of the method, the step of depleting miR-10b-5pfragments from the population of RNA molecules comprises:

-   -   adding miR-10b-5p specific oligonucleotide probes to a sample        containing the population of RNA molecules, wherein each        miR-10b-5p specific probe comprises a nucleotide sequence that        is the complement to a nucleotide sequence of miR-10b-5p;    -   forming a complex between one or more miR-10b-5p fragments and a        miR-10b-5p specific oligonucleotide probe; and    -   removing the miR-10b-5p:oligonucleotide complexes from the        sample.

Each miR-10b-5p specific oligonucleotide probe may comprise one ormultiple copies of a nucleotide sequence that is the complement to thenucleotide sequence of miR-10b-5p.

In another embodiment, the 5′end, the 3′end or both ends of eachmiR-10a-5p specific oligonucleotide probe or each miR-10b-5p specificoligonucleotide probe are modified, wherein the modification(s)facilitate the removal of the miR-10a-5p:oligonucleotide complexesand/or miR-10b-5p:oligonucleotide complexes from the sample.

In a further embodiment, each of the miR-10a-5p specific oligonucleotideprobes and/or the miR-10b-5p specific oligonucleotide probes has a 5′biotin modification, a 3′ biotin modification, a 5′ dioxigeninmodification, a 3′ dioxigenin modification, and/or a 5′ dinitrophenolmodification.

In another embodiment, the miR-10a-5p specific oligonucleotide probesand/or the miR-10b-5p specific oligonucleotide probes are immobilized ona solid support.

In another aspect, disclosed is a method of improving global geneexpression analysis for a population of RNA molecules derived from humanurine, the method comprising the step of blocking miR-10a-5p and/ormiR-10b-5p fragments in the population of RNA molecules. The method maycomprise blocking only miR-10a-5p fragments in the population of RNAmolecules. The method may comprise blocking only miR-10b-5p fragments inthe population of RNA molecules. The method may comprise blocking bothmiR-10a-5p and miR-10b-5p fragments in the population of RNA molecules.

In an embodiment of the method, the step of blocking the miR-10a-5pfragments in the population of RNA molecules comprises:

adding miR-10a-5p specific oligonucleotide probes to a sample containingthe population of RNA molecules, wherein each miR-10a-5p specificoligonucleotide probe comprises a nucleotide sequence that is thecomplement to a nucleotide sequence of the miR-10a-5p; and

-   -   forming a complex between one or more miR-10a-5p fragments and a        miR-10a-5p specific oligonucleotide probe.

In another embodiment, the step of blocking the miR-10b-5p fragments inthe population of RNA molecules comprises:

-   -   adding miR-10b-5p specific oligonucleotide probes to a sample        containing the population of RNA molecules, wherein each        miR-10b-5p specific oligonucleotide probe comprises a nucleotide        sequence that is the complement to a nucleotide sequence of        miR-10b-5p; and    -   forming a complex between one or more miR-10b-5p fragments and a        miR-10b-5p specific oligonucleotide probe.

In another embodiment, the 5′end, the 3′end or both ends of eachmiR-10a-5p specific oligonucleotide probe and/or miR-10b-5p specificoligonucleotide probe is modified to prevent ligation.

In a further embodiment, the 5′end or the 3′end of each miR-10a-5pspecific oligonucleotide probe and/or miR-10b-5p specificoligonucleotide probe is modified by incorporating a dideoxy nucleotide.

In a further embodiment, the 5′end or the 3′end of each miR-10a-5pspecific oligonucleotide probe and/or miR-10b-5p specificoligonucleotide probe is modified with biotin.

In a further embodiment, the 5′end of each miR-10a-5p specificoligonucleotide probe and/or miR-10b-5p specific oligonucleotide probeis modified with biotin and the 3′end of each miR-10a-5p specificoligonucleotide probe and/or miR-10b-5p specific oligonucleotide probeis modified by incorporating a dideoxy nucleotide.

In another embodiment of any of the methods described above, the globalgene expression analysis can be microarray analysis, libraryconstruction, reverse transcription, amplification, transcriptomeprofiling, expression analysis and/or sequencing. In a furtherembodiment, the sequencing is next generation sequencing.

In another aspect, disclosed is a method of performing next generationsequencing of a population of small RNA derived from human urine, themethod comprising:

-   -   adding miR-10a-5p specific oligonucleotide probes and/or        miR-10b-5p specific oligonucleotide probes to a sample        containing the population of RNA molecules, wherein each        miR-10a-5p specific oligonucleotide probe comprises a nucleotide        sequence that is the complement to a nucleotide sequence of        miR-10a-5p and wherein each miR-10b-5p specific oligonucleotide        probe comprises a nucleotide sequence that is the complement to        a nucleotide sequence of miR-10b-5p;    -   forming a complex between one or more miR-10a-5p fragments and a        miR-10a-5p specific oligonucleotide probe and/or forming a        complex between one or more miR-10b-5p fragments and a        miR-10b-5p specific oligonucleotide probe; and    -   removing the miR-10a-5p:oligonucleotide complexes and/or        miR-10b-5p:oligonucleotide complexes from the sample, wherein        the remaining sample contains a miR-10a-5p and/or miR-10b-5p        depleted population of small RNA molecules;    -   preparing a library using the remaining sample; and    -   sequencing the library.

In one embodiment, only miR-10a-5p specific oligonucleotide probes areadded and the remaining sample contains a miR-10a-5p depleted populationof small RNA molecules. In another embodiment, only miR-10b-5p specificoligonucleotide probes are added and the remaining sample contains amiR-10b-5p depleted population of small RNA molecules. In a furtherembodiment, miR-10a-5p specific oligonucleotide probes and miR-10b-5pspecific oligonucleotide probes are added and the remaining samplecontains a miR-10a-5p and miR-10b-5p depleted population of small RNAmolecules.

In another embodiment, each miR-10a-5p specific oligonucleotide probecomprises one or multiple copies of a nucleotide sequence that is thecomplement to the nucleotide sequence of the miR-10a-5p.

In another embodiment, each miR-10b-5p specific oligonucleotide probecomprises one or multiple copies of a nucleotide sequence that is thecomplement to the nucleotide sequence of miR-10b-5p.

In another embodiment, the miR-10a-5p:oligonucleotide complexes and/ormiR-10b-5p:oligonucleotide complexes are removed by size exclusionchromatography.

In another embodiment, the miR-10a-5p:oligonucleotide complexes and/ormiR-10b-5p:oligonucleotide complexes are removed by using siliconcarbide.

In a further embodiment, the step of removing themiR-10a-5p:oligonucleotide complexes and/or miR-10b-5p:oligonucleotidecomplexes from the sample comprises:

-   -   combining the sample with a binding buffer, an alcohol and a        silicon carbide slurry to provide a binding mixture, wherein the        alcohol concentration of the binding mixture is about 1-30%        (v/v) to affect selective binding of the miR-10a-5p :        oligonucleotide complexes and/or miR-10b-5p:oligonucleotide        complexes to the silicon carbide;    -   removing the miR-10a-5p:oligonucleotide complexes and/or        miR-10b-5p:oligonucleotide complexes bound to SiC from the        sample; and    -   collecting the remaining sample containing the miR-10a-5p and/or        miR-10b-5p depleted population of small RNA molecules.

In further embodiment, the step of removing themiR-10a-5p:oligonucleotide and/or miR-10b-5p:oligonucleotide complexescomprises:

-   -   combining the sample with a binding buffer and alcohol to        provide a binding mixture;    -   applying the binding mixture to a silicon carbide column,        wherein the alcohol concentration of the binding mixture is        about 1-30% (v/v) to affect selective binding of the        miR-10a-5p:oligonucleotide complexes and/or        miR-10b-5p:oligonucleotide complexes to the silicon carbide;    -   collecting the column flowthrough containing the miR-10a-5p        and/or miR-10b-5p depleted population of small RNA molecules.

The alcohol concentration of the binding mixture can be about 1-10%(v/v). In a further embodiment, the alcohol is ethanol.

In another embodiment, the 5′end, the 3′end or both ends of eachmiR-10a-5p specific oligonucleotide probe and/or miR-10b-5p specificoligonucleotide probe is modified and wherein themiR-10a-5p:oligonucleotide complexes and/or miR-10b-5p:oligonucleotidecomplexes are removed by:

-   -   selectively binding the miR-10a-5p:oligonucleotide complexes        and/or miR-10b-5p:oligonucleotide complexes to a solid support        comprising a protein or antibody that specifically interacts        with an end modification on the miR-10a-5p specific        oligonucleotide probe and/or miR-10b-5p specific oligonucleotide        probe; and    -   collecting an unbound fraction of the sample containing the        miR-10a-5p and/or miR-10b-5p depleted population of small RNA        molecules.

In a further embodiment, the 5′end or the 3′end of each miR-10a-5pspecific oligonucleotide probe and/or miR-10b-5p specificoligonucleotide probe is modified with biotin and the solid supportcomprises avidin or streptavidin.

In a further embodiment, the 5′end or the 3′end of each miR-10a-5pspecific oligonucleotide probe and/or miR-10b-5p specificoligonucleotide probe is modified with digoxigenin and the solid supportcomprises digoxigenin specific antibodies.

In a further embodiment, the 5′end of each miR-10a-5p specificoligonucleotide probe and/or miR-10b-5p specific oligonucleotide probeis modified with dinitrophenol and the solid support comprisesdinitrophenol specific antibodies.

In a further embodiment, the solid support comprises polymeric beads,which may be magnetic or non-magnetic.

In another aspect, disclosed is a method of performing next generationsequencing of a population of small RNA derived from human urine, themethod comprising:

-   -   adding miR-10a-5p specific oligonucleotide probes and/or        miR-10b-5p specific oligonucleotide probes to a sample        containing the population of RNA molecules, wherein each        miR-10a-5p specific oligonucleotide probe comprises a nucleotide        sequence that is the complement to a nucleotide sequence of        miR-10a-5p and wherein each miR-10b-5p specific oligonucleotide        probe comprises a nucleotide sequence that is the complement to        a nucleotide sequence of miR-10b-5p;    -   forming a complex between one or more miR-10a-5p fragments and a        miR-10a-5p specific oligonucleotide probe and/or forming a        complex between one or more miR-10b-5p fragments and a        miR-10b-5p specific oligonucleotide probe to provide a        miR-10a-5p and/or miR-10b-5p blocked sample;    -   preparing a library using the miR-10a-5p and/or miR-10b-5p        blocked sample; and    -   sequencing the library.

In one embodiment, the 5′end, the 3′end or both ends of each miR-10a-5pspecific oligonucleotide probe and/or miR-10b-5p specificoligonucleotide probe is modified to prevent ligation.

In a further embodiment, the 5′end or the 3′end of each miR-10a-5pspecific oligonucleotide probe and/or miR-10b-5p specificoligonucleotide probe is modified by incorporating a dideoxy nucleotide.

In a further embodiment, the 5′end or the 3′end of each miR-10a-5pspecific oligonucleotide probe and/or miR-10b-5p specificoligonucleotide probe is modified with biotin.

In a further embodiment, the 5′end of each miR-10a-5p specificoligonucleotide probe and/or miR-10b-5p specific oligonucleotide probeis modified with biotin and the 3′end of each miR-10a-5p specificoligonucleotide probe and/or miR-10b-5p specific oligonucleotide probeis modified by incorporating a dideoxy nucleotide.

In a further aspect, disclosed are kits that are useful for improvingglobal gene expression analysis for a population of RNA moleculesderived from human urine. The kit comprises one or more miR-10a-5pspecific oligonucleotide probes, wherein each miR-10a-5p specificoligonucleotide probe comprises a nucleotide sequence that is thecomplement to a nucleotide sequence of miR-10a-5p and/or, one or moremiR-10b-5p specific oligonucleotide probes, wherein each miR-10b-5pspecific oligonucleotide probe comprises a nucleotide sequence that isthe complement to a nucleotide sequence of miR-10b-5p.

In one embodiment, the 5′end, the 3′end or both ends of each miR-10a-5pspecific oligonucleotide probe and/or miR-10b-5p specificoligonucleotide probe is modified.

In a further embodiment, the 5′end or the 3′end of each miR-10a-5pspecific oligonucleotide probe and/or miR-10b-5p specificoligonucleotide probe is modified with biotin.

In a further embodiment, the 5′end or the 3′end of each miR-10a-5pspecific oligonucleotide probe and/or miR-10b-5p specificoligonucleotide probe is modified with digoxigenin.

In a further embodiment, the 5′end of each miR-10a-5p specificoligonucleotide probe and/or miR-10b-5p specific oligonucleotide probeis modified with dinitrophenol.

In a further embodiment, the 3′end of each miR-10a-5p specificoligonucleotide probe and/or miR-10b-5p specific oligonucleotide probeis modified by incorporating a dideoxy nucleotide.

In a further embodiment, the 5′end of each miR-10a-5p specificoligonucleotide probe and/or miR-10b-5p specific oligonucleotide probeis modified with biotin and the 3′end of each miR-10a-5p specificoligonucleotide probe and/or miR-10b-5p specific oligonucleotide probeis modified by incorporating a dideoxy nucleotide.

In a further embodiment, the miR-10a-5p specific oligonucleotide probeand/or miR-10b-5p specific oligonucleotide probe is immobilized on asolid support.

In an embodiment of any of the methods or kits described above, thenucleotide sequence of miR-10a-5p has at least 90% sequence identity tothe nucleotide sequence of SEQ ID NO: 1. In a further embodiment, thenucleotide sequence of miR-10a-5p comprises the nucleotide sequence ofSEQ ID NO: 1.

In another embodiment of any of the methods or kits described above, thenucleotide sequence of miR-10b-5p has at least 90% sequence identity tothe nucleotide sequence of SEQ ID NO: 3. In a further embodiment, thenucleotide sequence of miR-10b-5p comprises the nucleotide sequence ofSEQ ID NO: 3.

In another embodiment of any of the methods or kits described above, themiR-10a-5p specific oligonucleotide probe has at least 90% sequenceidentity to the nucleotide sequence of SEQ ID NO: 2. In a furtherembodiment, the miR-10a-5p specific oligonucleotide probe comprises thenucleotide sequence of SEQ ID NO: 2.

In another embodiment of any of the methods or kits described above, themiR-10b-5p specific oligonucleotide probe has at least 90% sequenceidentity to the nucleotide sequence of SEQ ID NO: 4. In a furtherembodiment, the miR-10b-5p specific oligonucleotide probe comprises thenucleotide sequence of SEQ ID NO: 4.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph that shows the number of read per million (RPM) mappedmiRNAs over all clipped reads for a control (non-depleted) urine RNAsample and miR-10a-5p fragment-depleted urine RNA samples from healthydonors.

FIG. 2 is a graph that shows the correspondence between insert size andthe overall % of reads for a control (non-depleted) urine RNA samplesand miR-10a-5p fragment-depleted urine RNA sample from healthy donors.

FIG. 3 is a graph that shows the number of read per million (RPM) mappedmiRNAs over all clipped reads for a control (non-depleted) urine RNAsample and miR-10b-5p fragment-depleted urine RNA samples from healthydonors.

FIG. 4 is a graph that shows the correspondence between insert size andthe overall % of reads for a control (non-depleted) urine RNA sample andmiR-10b-5p fragment-depleted urine RNA samples from healthy donors.

FIG. 5 is a graph depicting the average number of miRNA detected in NGSruns from libraries created from both control (non-depleted) urine RNA,as well as the miR-10b-5p fragment-depleted urine RNA samples.

FIG. 6 is a graph of insert sizes that shows the correspondence betweeninsert size and the overall % of reads for a control (non-depleted)urine RNA sample and both miR-10a-5p and miR-10b-5p fragments-depletedurine RNA samples from healthy donors.

FIG. 7 is a graph depicting the average number of miRNA detected in NGSruns from libraries created from both control (non-depleted) urine RNA,as well as both miR-10a-5p and miR-10b-5p fragments-depleted urine RNAsamples.

DESCRIPTION

It has now been demonstrated that the disproportionate abundance ofmiR-10a-5p and miR-10b-5p fragments in human urine RNA samples poses amajor impediment to accurate detection and quantification of other,better characterized and/or more diagnostically relevant miRNAs. Thechallenges experienced in the generation of a global expression profileof miRNA in urine—due to the sheer amount of miR-10a-5p and miR-10b-5ppresent in human urine—are well exemplified in global gene expressionanalysis employing next generation sequencing (NGS).

There are many different platforms that can be used for NGS of smallRNA, including Roche 454, Roche GS FLX Titanium, Illumina MiSeq,Illumina HiSeq, Illumina Genome Analyzer IIX, Illumina MiniSeq, IlluminaNextSeq, Illumina NovaSeq Life Technologies SOLiD4, Life TechnologiesIon Proton, Complete Genomics, Helicos Biosciences Heliscope, andPacific Biosciences SMRT. All of these different platforms follow thesame general procedure for NGS of small RNA. Namely, a DNA sequencinglibrary is prepared using purified RNA. Library preparation includestranscribing the RNA into cDNA, ligating the cDNA molecules with 5′ and3′ adaptors, and amplifying the ligated DNA fragments. These relativelyshort DNA fragments are then massively parallel sequenced andbioinformatics analysis applied to de-multiplex samples, align, annotateand aggregate reads.

Within the total population of miRNAs present in human urine, it hasbeen found that certain miRNAs are disproportionately abundant. Two ofthe most overrepresented miRNAs are miR-10a-5p and miR-10b-5p, which canaccount for over 50% of the miRNA present in in human urine (El-Mogy etal., 2018). These miRNAs have been extensively studied in manybiological pathways, including cancer development and progression(Veerla et al., 2009; Xiao et al., 2014; Ma et al., 2015; Zhang et al.,2015; Abdelmaksoud-Dammak et al., 2017; Arai et al., 2017). It has nowbeen demonstrated that the disproportionate abundance of miR-10a-5p andmiR-10b-5p in human urine RNA samples poses a major impediment toaccurate detection and quantification of other, less abundant and/orpotentially predictive miRNAs. The challenges experienced in thegeneration of a global expression profile of miRNA in urine—due to thesheer amount of miR-10a-5p and miR-10b-5p present in the miRNA found inhuman urine—are well exemplified in global gene expression analysisemploying NGS.

The number of times each sequence in the library has been “read” (e.g.sequenced) is of utmost importance in determining both how reliably itcan be called and its abundance relative to other sequences in the samesample. As miR-10a-5p and miR-10b-5p are of the most abundant miRNAsequences in human urine, the greatest proportion of miRNA reads in anygiven small RNA library—prepared from RNA purified from human urine—ismapped to these two miRNAs (Zhou et al., 2017). In the case of globalmiRNA expression analysis, this produces much less reliable data forrelatively rare miRNA transcripts, which may be read at the level of“noise”, or not called at all, because they constitute a very smallproportion of miRNAs at the outset. The total number of all reads forany sample on any NGS platform is finite. As such, a much smallerproportion of reads is allocated for all the other miRNAs present, whichmay be of a much greater interest than miR-10a-5p and miR-10b-5p. Thesequencing of the library will be skewed towards the sequencing of theseoverly abundant miR-10a-5p and miR-10b-5p fragments.

It has now been surprisingly found that global gene expression analysisfor small RNA samples derived from human urine samples can be improvedby selectively depleting the abundant miR-10a-5p and/or miR-10b-5pfragments prior to library preparation or by selectively blocking themiR-10a-5p and/or miR-10b-5p fragments in the RNA samples to preventthem from acting as a substrate during library preparation. Byselectively depleting or blocking the miR-10a-5p and/or miR-10b-5pfragments, it is possible to improve the ratio of useful data (e.g. datamapped to less abundant miRNAs of interest) to non-useful data (e.g.data mapped to miR-10a-5p or miR-10b-5p fragments). As a result, globalgene expression analysis can be improved, for example, by increasing thesensitivity of the global gene expression analysis (e.g. reduction ofbackground noise) and by increasing the reliability of the obtainedexpression data. This can be beneficial when performing research anddiscovery of novel miRNA markers in human urine, as well as studies thatrely on the ability to see changes in low expressing but significantmiRNA.

Method of Improving Global Gene Expression Analysis of Human UrineDerived RNA

Disclosed is a method of improving global gene expression analysis for apopulation of RNA molecules derived from human urine.

As used herein, “global gene expression analysis” includes anyquantitative method for investigating a population of RNA species. Inthe disclosed method, the population of RNA species are derived fromhuman urine. Global gene expression analysis can be carried out, forexample, by way of microarray analysis, library construction, reversetranscription, amplification, transcriptome profiling, expressionanalysis and sequencing, including next generation sequencing.

Selectively depleting or blocking miR-10a-5p and/or miR-10b-5p fragmentspresent in the population of RNA molecules can improve global geneexpression analysis in a population of RNA molecules derived from humanurine. By selectively depleting or blocking the miR-10a-5p and/ormiR-10b-5p fragments present in the population of RNA molecules, theratio of useful data (e.g. data mapped to miRNAs of interest) tonon-useful data (e.g. data mapped to miR-10a-5p or miR-10b-5p fragments)obtained by the global gene expression analysis is improved.

One embodiment of the disclosed method of improving global geneexpression analysis for a population of RNA molecules derived from humanurine comprises selectively depleting or blocking miR-10a-5p fragmentspresent in the population of RNA molecules. In another embodiment, themethod comprises selectively depleting or blocking miR-10b-5p fragmentspresent in the population of RNA molecules. In a further embodiment, themethod comprises selectively depleting or blocking miR-10a-5p fragmentsand miR-10b-5p fragments present in the population of RNA molecules.

Human Urine Derived RNA Molecules

The disclosed method for improving global gene expression can beperformed using an initial population of RNA molecules, which is totalRNA isolated from human urine. Urine samples can be collected and storedusing conventional methods known in the art. It may be desirable toemploy urine collection tubes that prevent RNA degradation, such as, butnot limited to Norgen's Urine Collection and Preservation Tubes (Cat#18116, 18120, 18111, Norgen Biotek Corp., Thorold, Canada). Methods forthe isolation of total RNA from human urine are well known in the art.Suitable methods for the isolation of total RNA include but are notlimited to the use of phenol/chloroform, the use of silicon carbide(SiC), the use of silica, and alcohol precipitation.

The initial population of RNA molecules can also be small RNA isolatedfrom human urine. Again, suitable methods for the isolation of small RNAare known in the art. Suitable methods include, but are not limited, tothe use of phenol/chloroform, the use of silicon carbide, and the use ofsilica. In a preferred embodiment, the initial population of RNAmolecules is small RNA isolated from human urine samples using SiC.

Selective Depletion of miR-10a-5p and/or miR-10b-5p Fragments

In one embodiment, disclosed is a method of improving global geneexpression analysis for a population of RNA molecules derived from humanurine, wherein the method comprises the step of depleting miR-10a-5pfragments from the population of RNA molecules. It will be appreciatedthat the disclosed method does not require the complete removal of allmiR-10a-5p fragments.

The resulting population of RNA molecules that are depleted ofmiR-10a-5p fragments can be used in downstream global gene expressionanalysis applications. This method is particularly suitable forpreparing small RNA for next generation sequencing applications. Byremoving the highly abundant miR-10a-5p fragments prior to preparationof the sequencing library, the signal to noise ratio can be improved.

miR-10a-5p fragments can be depleted from the population of RNAmolecules by selectively removing the fragments. In one embodiment,miR-10a-5p fragments are selectively removed by:

-   -   adding miR-10a-5p specific oligonucleotide probes to a sample        containing the population of RNA molecules, wherein each        miR-10a-5p specific oligonucleotide probe comprises a nucleotide        sequence that is the complement to a nucleotide sequence of        miR-10a-5p;    -   forming a complex between one or more miR-10a-5p fragments and a        miR-10a-5p specific oligonucleotide probe; and    -   removing the miR-10a-5p:oligonucleotide complexes from the        sample.

The miR-10a-5p specific oligonucleotide probes are designed to becomplementary to miR-10a-5p, and thus are capable of hybridizing withthe miR-10a-5p fragments. The miR-10a-5p specific oligonucleotide probecan be various lengths, so long as it contains sufficient bases to allowthe probe to specifically bind to the miR-10a-5p fragments. ThemiR-10a-5p specific oligonucleotide probe may be 6-200 bases and morepreferably 20-50 bases.

In a more preferred embodiment, the miR-10a-5p specific oligonucleotideprobe is designed to be the complement of the 23 base miR-10a-5pfragment with the sequence:

(SEQ ID NO: 1) 5′- UACCCUGUAGAUCCGAAUUUGUG -3′.

In this embodiment, the miR-10a-5p specific oligonucleotide probe cancomprise the following sequence:

(SEQ ID NO: 2) 5′- CACAAATTCGGATCTACAGGGTA -3′.

In further preferred embodiments, the miR-10a-5p specificoligonucleotide probe can be designed to be the complement of anucleotide having at least 90%, 95% or 99% identity with the nucleotidesequence of SEQ ID NO:1. The miR-10a-5p specific oligonucleotide probecan comprise a nucleotide sequence having at least 90%, 95% or 99%identity with the nucleotide sequence of SEQ ID NO:2.

In a further embodiment, the miR-10a-5p specific oligonucleotide probemay comprise one or multiple copies of the complement to the miR-10a-5pfragment. The miR-10a-5p specific oligonucleotide probe may comprise2-20 copies of the complement to the miR-10a-5p fragment, and morepreferably comprises 7 copies of the complement to the miR-10a-5pfragment.

After hybridization, the miR-10a-5p:oligonucleotide complexes areremoved from the RNA sample to provide the miR-10a-5p depletedpopulation of RNA molecules. A variety of different methods can beemployed to remove the miR-10a-5p:oligonucleotide complexes from the RNAsample.

In one embodiment, the miR-10a-5p specific oligonucleotide probeincludes modifications to facilitate the use of solid supports for theselective removal of the miR-10a-5p:oligonucleotide complexes. Forexample, the miR-10a-5p specific oligonucleotide probe may include a5′end modification, a 3′end modification, an internal modification orcombination thereof, that allows the miR-10a-5p:oligonucleotidecomplexes to covalently or non-covalently bind to a solid support, whichcomprises a functional group, a protein or an antibody, whichspecifically interacts with the modification. For example, theoligonucleotide probe can be provided with a 5′ or 3′ biotinmodification for selective binding to solid supports comprising avidinor streptavidin. The oligonucleotide probe can be provided with a 5′ or3′ digoxigenin modification for selective binding to solid supportscomprising digoxigenin specific antibodies. The oligonucleotide probecan be provided with a 5′ dinitrophenol modification for selectivebinding to solid supports comprising dinitrophenol specific antibodies.Examples of solid supports that may be used to selectively removemiR-10a-5p:oligonucleotide complexes include resin packed columns andpurification beads, which may be magnetic or non-magnetic (such aspolystyrene).

In a preferred embodiment, the miR-10a-5p:oligonucleotide complexes areremoved by:

selectively binding the miR-10a-5p:oligonucleotide complexes to a solidsupport comprising a protein or antibody that specifically interactswith an end modification on the oligonucleotide probe; and

-   -   collecting an unbound fraction of the sample containing the        miR-10a-5p depleted population of small RNA molecules.

The miR-10a-5p specific oligonucleotide probe preferably comprises a5′end or a 3′end biotin modification and the solid support preferablycomprises magnetic beads that are coupled to avidin or streptavidin. Ina further preferred embodiment, the magnetic beads are coupled tostreptavidin. Following selectively binding of themiR-10a-5p:oligonucleotide complexes to the magnetic beads, the boundmagnetic beads can be removed from the RNA sample using a magnet,thereby removing the miR-10a-5p:oligonucleotide complex from the RNAsample. The unbound fraction of RNA sample containing the miR-10a-5pdepleted population of small RNA molecules can then be collected for usein downstream global gene expression analysis applications.

In another embodiment, the miR-10a-5p specific oligonucleotide probescan be immobilized onto a solid support. In this embodiment, the RNAsample containing the population of RNA molecules can be added to thesolid support or vice versa. miR-10a-5p fragments will hybridize to theoligonucleotide probes immobilized on the solid support. The unboundfraction of the sample containing the miR-10a-5p depleted population ofsmall RNA molecules can then be collected for use in downstream globalgene expression analysis applications.

In another embodiment, the miR-10a-5p:oligonucleotide complexes can beremoved from the RNA sample using size exclusion chromatography, whichis based on the differential binding of molecules to a matrix based onsize. In a preferred embodiment, silica columns can be used to separatethe miR-10a-5p:oligonucleotide complexes from the mixture.

In a further embodiment, the miR-10a-5p:oligonucleotide complexes can beremoved from the RNA sample using a size selective isolation methodemploying SiC. The RNA sample containing the miR-10a-5p:oligonucleotidecomplexes can be combined with a binding buffer, an alcohol and SiC toprovide a binding mixture. The alcohol concentration of the bindingmixture is adjusted to determine the cut-off size of RNA molecules thatwill be preferentially bound to the SiC. By using a lower alcoholconcentration, the larger miR-10a-5p:oligonucleotide complexes containedin the RNA sample will selectively bind to the SiC, whereas the smallermiRNAs will remain in the liquid phase.

The alcohol concentration in the binding mixture can be adjusted usingany alcohol known in the art. Examples of suitable alcohols include arebut not limited to ethanol, isopropanol and methanol. To achieve sizeselective binding of the miR-10a-5p:oligonucleotide complexes to theSiC, the alcohol concentration of the binding mixture can preferably beadjusted with ethanol to a concentration of between 1-30% (v/v), andmore preferably between 1-10% (v/v).

The size selective isolation method can be performed using a SiC slurryor a SiC column. In either embodiment, the size selective binding stepcan be performed under low salt conditions and slightly acidic toneutral pH conditions of about pH 4-7. The largermiR-10a-5p:oligonucleotide complexes contained in the RNA sample willcome into contact with the SiC and selectively bind to the SiCparticles. The unbound small miRNAs will remain in the liquid phase. Inembodiments employing SiC in a slurry format, the liquid phasecontaining the small miRNAs can be collected, for example, by pelletingthe SiC through centrifugation and decanting the liquid phase containingthe small miRNAs. For embodiments using a SiC column, such as a spincolumn, the larger miR-10a-5p:oligonucleotide complexes selectivelybound to the SiC will be retained in the column and the flowthroughcollected. The collected small miRNAs can be used in downstream globalgene expression analysis.

In a further embodiment, disclosed is a method of improving global geneexpression analysis for a population of RNA molecules derived from humanurine, wherein the method comprises the step of depleting miR-10b-5pfragments from the population of RNA molecules. It will be appreciatedthat the disclosed method does not require the complete removal of allmiR-10b-5p fragments

The miR-10b-5p fragments can be depleted from the population of RNAmolecules by selectively removing the fragments. In one embodiment,miR-10b-5p fragments are selectively removed by:

-   -   adding miR-10b-5p specific oligonucleotide probes to a sample        containing the population of RNA molecules, wherein each probe        comprises a nucleotide sequence that is the complement to the        nucleotide sequence of miR-10b-5p; forming a complex between one        or more miR-10b-5p fragments and a miR-10b-5p specific        oligonucleotide probe; and    -   removing the miR-10b-5p:oligonucleotide complexes from the        sample.

The miR-10b-5p specific oligonucleotide probe can be various lengths, solong as it contains sufficient bases to allow the probe to specificallybind to the miR-10b-5p fragments. The oligonucleotide probe may be 6-200bases and more preferably 20-50 bases.

In a more preferred embodiment, the oligonucleotide probe is designed tobe the complement of the 23 base miR-10b-5p fragment having thesequence:

(SEQ ID NO: 3) 5′- UACCCUGUAGAACCGAAUUUGUG -3′.

In this embodiment, the oligonucleotide probe can comprise the followingsequence:

(SEQ ID NO: 4) 5′- CACAAATTCGGTTCTACAGGGTA -3′.

In further preferred embodiments, the miR-10b-5p specificoligonucleotide probe can be designed to be the complement of anucleotide having at least 90%, 95% or 99% identity with the nucleotidesequence of SEQ ID NO:3. The miR-10b-5p specific oligonucleotide probecan comprise a nucleotide sequence having at least 90%, 95% or 99%identity with the nucleotide sequence of SEQ ID NO:4.

In a further embodiment, the oligonucleotide probe may comprise one ormultiple copies of the complement to the miR-10b-5p fragment. ThemiR-10b-5p specific oligonucleotide probe may comprise 2-20 copies ofthe complement to the miR-10b-5p fragment, and more preferably comprises7 copies of the complement to the miR-10b-5p fragment.

After hybridization, the miR-10b-5p:oligonucleotide complexes areremoved from the RNA sample to provide the miR-10b-5p depletedpopulation of RNA molecules. A variety of different methods can beemployed to remove the miR-10b-5p:oligonucleotide complexes from the RNAsample including selective binding to a solid support and size selectiveisolation using a SiC slurry or a SiC column as described above. It willbe apparent to the skilled person that the methods described herein forthe removal of miR-10a-5p:oligonucleotide complexes can be adapted forthe removal of miR-10b-5p:oligonucleotide complexes, for example,through the use of modified miR-10b-5p specific oligonucleotide probes.

The disclosed method of improving global gene expression analysis for apopulation of RNA molecules derived from human urine may comprise theselective depletion of miR-10a-5p fragments or the selective depletionmiR-10b-5p fragments. In alternate embodiments, the method may comprisethe selective depletion of miR-10a-5p fragments and miR-10b-5pfragments, wherein the step of depleting the miR-10a-5p fragments andthe step of depleting the miR-10b-5p fragments are carried outsuccessively or concurrently.

Selective Blocking of miR-10a-5p and/or miR-10b-5p Fragments

In another embodiment, disclosed is a method of improving global geneexpression analysis for a population of RNA molecules derived from humanurine, wherein the method comprises the step of selectively blockingmiR-10a-5p fragments in the population of RNA molecules.

As used herein, “selectively blocking miR-10a-5p fragments” refers toany modification that renders the miR-10a-5p fragments an unsuitablesubstrate in a downstream global gene expression analysis application.For example, the miR-10a-5p fragments can be blocked by hybridizing themiR-10a-5p fragments with a miR-10a-5p specific oligonucleotide probehaving a complementary sequence to form miR-10a-5p:oligonucleotidecomplexes.

The resulting population of RNA molecules including the blockedmiR-10a-5p fragments can be used in downstream global gene expressionanalysis applications. This method is particularly suitable forpreparing small RNA for next generation sequencing applications in orderto improve the signal to noise ratio. By blocking the highly abundantmiR-10a-5p fragments (e.g. by forming double stranded DNA-RNA hybridswith the miR-10a-5p specific oligonucleotide probes), these fragmentswill no longer be a suitable substrate for any of the steps in librarypreparation, including the initial attachment of the 5′ and 3′ adaptors.

The miR-10a-5p fragments can be selectively blocked in a population ofRNA molecules by:

-   -   adding miR-10a-5p specific oligonucleotide probes to a sample        containing the population of RNA molecules, wherein each        miR-10a-5p specific oligonucleotide probe comprises a nucleotide        sequence that is the complement to a nucleotide sequence of        miR-10a-5p; and    -   forming a complex between one or more miR-10a-5p fragments and a        miR-10a-5p specific oligonucleotide probe.

Any of the miR-10a-5p specific oligonucleotide probes described abovecan be used to selectively block the miR-10a-5p fragments contained inthe RNA sample by forming a complex between the miR-10a-5p fragments andthe miR-10a-5p specific oligonucleotide probe.

In a further embodiment, the 5′end, the 3′ end or both ends of themiR-10a-5p specific oligonucleotide probe is modified to preventligation. The 5′end of the miR-10a-5p specific oligonucleotide probe canbe selectively blocked through the use of inverted dideoxy-T, the use ofdephoshorylated 5′ ends, the use of biotin and any other suitable 5′ endmodification method. The 3′ end of the miR-10a-5p specificoligonucleotide probe can also be blocked using a suitable 3′endmodification method, including but not limited to, the use of inverteddT, dideoxy-C, and other dideoxy nucleotides.

In a preferred embodiment, the miR-10a-5p specific oligonucleotide probeis blocked at both the 5′ and 3′ end, thereby preventing the attachmentof 5′ and 3′ adaptors to the miR-10a-5p specific oligonucleotide probe.By blocking one or both ends of the miR-10a-5p specific oligonucleotideprobe, it is possible to avoid the miR-10a-5p specific oligonucleotideprobes themselves being incorporated into the sequence library andcontributing to the background noise. In a preferred embodiment, themiR-10a-5p specific oligonucleotide probe is blocked using a biotin atthe 5′ end and using a dideoxy base at the 3′ end.

In another embodiment, disclosed is a method of improving global geneexpression analysis for a population of RNA molecules derived from humanurine, wherein the method comprises the step of selectively blockingmiR-10b-5p fragments in the population of RNA molecules.

As used herein, “selectively blocking miR-10b-5p fragments” refers toany modification that renders the miR-10b-5p fragments an unsuitablesubstrate in a downstream global gene expression analysis application.For example, the miR-10b-5p fragments can be blocked by hybridizing themiR-10b-5p fragments with a miR-10b-5p specific oligonucleotide probehaving a complementary sequence to form miR-10b-5p:oligonucleotidecomplexes.

The resulting population of RNA molecules including the blockedmiR-10b-5p fragments can also be used in downstream global geneexpression analysis applications. This method is also particularlysuitable for preparing small RNA for next generation sequencingapplications in order to improve the signal to noise ratio. By blockingthe highly abundant miR-10b-5p fragments (e.g. by forming doublestranded DNA-RNA hybrids with the miR-10b-5p specific oligonucleotideprobes), these fragments will no longer be a suitable substrate for anyof the steps in library preparation, including the initial attachment ofthe 5′ and 3′ adaptors.

The miR-10b-5p fragments can be selectively blocked in a population ofRNA molecules by:

-   -   adding miR-10b-5p specific oligonucleotide probes to a sample        containing the population of RNA molecules, wherein each        miR-10b-5p specific oligonucleotide probe comprises a nucleotide        sequence that is the complement to a nucleotide sequence of        miR-10b-5p; and    -   forming a complex between one or more miR-10b-5p fragments and a        miR-10b-5p specific oligonucleotide probe.

Any of the miR-10b-5p specific oligonucleotide probes described abovecan be used to selectively block the miR-10b-5p fragments contained inthe RNA sample by forming a complex between the miR-10b-5p fragments andthe miR-10b-5p specific oligonucleotide probe.

In a further embodiment, the 5′end, the 3′end or both ends of themiR-10b-5p specific oligonucleotide probe are modified to preventligation. The miR-10b-5p specific oligonucleotide probe can be similarlymodified as described above for modified miR-10a-5p specificoligonucleotide probes.

The disclosed method may comprise selectively blocking of miR-10a-5pfragments or selectively blocking miR-10b-5p fragments. In alternateembodiments, the method may comprise selectively blocking of miR-10a-5pfragments and miR-10b-5p fragments, wherein the step of blockingmiR-10a-5p fragments and the step of blocking miR-10b-5p fragments arecarried out successively or concurrently.

Next Generation Sequencing and Small RNA Libraries

Further disclosed is a method of performing next generation sequencingof a population of small RNA derived from human urine. In oneembodiment, the method comprises the provision of a miR-10a-5p fragmentdepleted population of small RNA molecules, which is then used toprepare the sequencing library. By removing the highly abundantmiR-10a-5p fragments prior to the preparation of the sequencing library,the sequencing capacity for the less abundant small RNA species isincreased and the efficiency of the sequencing reaction improved. Fewerresources are wasted during both the library preparation and sequencingsteps since the depleted miR-10a-5p fragments will not form part of thesequencing library and will therefore not be read during the sequencing.

In a preferred embodiment, the method of performing next generationsequencing of a population of small RNA derived from human urinecomprises:

-   -   adding miR-10a-5p specific oligonucleotide probes to a sample        containing the population of RNA molecules, wherein each probe        comprises a nucleotide sequence that is the complement to a        nucleotide sequence of miR-10a-5p;    -   forming a complex between one or more miR-10a-5p fragments and a        miR-10a-5p specific oligonucleotide probe; and    -   removing the miR-10a-5p:oligonucleotide complexes from the        sample, wherein the remaining sample contains a miR-10a-5p        depleted population of small RNA molecules;    -   preparing a library using the remaining sample; and sequencing        the library.

The miR-10a-5p depletion steps can be performed as described in greaterdetail above. The library preparation steps and sequencing steps can beperformed in accordance with known NGS protocols.

In another embodiment, the method of performing next generationsequencing of a population of small RNA derived from human urinecomprises blocking the miR-10a-5p fragments contained in the populationof RNA molecules to be sequenced, prior to the preparation of thelibrary. By blocking the miR-10a-5p fragments before the library isgenerated, the sequencing capacity for the less abundant small RNAspecies is increased and the efficiency of the sequencing reactionimproved. Fewer resources are wasted during both the library preparationand sequencing steps since the blocked miR-10a-5p fragments will not actas a substrate during the library preparation and will therefore not beread during the sequencing. Further, by using 5′end and/or 3′endmodified miR-10a-5p specific oligonucleotide probes which are themselvesblocked, incorporation of the oligonucleotide probes into the sequencelibrary and the consequential increase in background noise can beavoided.

In a preferred embodiment, disclosed is a method of performing nextgeneration sequencing of small RNA from a sample, comprising:

-   -   adding miR-10a-5p specific oligonucleotide probes to a sample        containing the population of RNA molecules, wherein each        miR-10a-5p specific oligonucleotide probe comprises a nucleotide        sequence that is the complement to a nucleotide sequence of        miR-10a-5p; and    -   forming a complex between one or more miR-10a-5p fragments and a        miR-10a-5p specific oligonucleotide probe to provide a        miR-10a-5p blocked sample;    -   preparing a library using the miR-10a-5p blocked sample; and    -   sequencing the library.

The miR-10a-5p blocking steps can be performed as described in greaterdetail above. In a further embodiment, the miR-10a-5p specificoligonucleotide probe can be modified at the 5′end, the 3′ end or atboth ends as described above in greater detail.

The library preparation steps and sequencing steps can be performed inaccordance with known NGS protocols.

In another embodiment, the method comprises the provision of amiR-10b-5p fragment depleted population of small RNA molecules, which isthen used to prepare the sequencing library. By removing the highlyabundant miR-10b-5p fragments prior to the preparation of the sequencinglibrary, the sequencing capacity for the less abundant small RNA speciesis increased and the efficiency of the sequencing reaction improved.Fewer resources are wasted during both the library preparation andsequencing steps since the depleted miR-10b-5p fragments will not formpart of the sequencing library and will therefore not be read during thesequencing.

In a preferred embodiment, the method of performing next generationsequencing of a population of small RNA derived from human urinecomprises:

-   -   adding miR-10b-5p specific oligonucleotide probes to a sample        containing the population of RNA molecules, wherein each probe        comprises a nucleotide sequence that is the complement to a        nucleotide sequence of miR-10b-5p;    -   forming a complex between one or more miR-10b-5p fragments and a        miR-10b-5p specific oligonucleotide probe; and    -   removing the miR-10b-5p:oligonucleotide complexes from the        sample, wherein the remaining sample contains a miR-10b-5p        depleted population of small RNA molecules;    -   preparing a library using the remaining sample; and    -   sequencing the library.

The miR-10b-5p depletion steps can be performed as described in greaterdetail above. The library preparation steps and sequencing steps can beperformed in accordance with known NGS protocols.

In another embodiment, the method of performing next generationsequencing of a population of small RNA derived from human urinecomprises blocking the miR-10b-5p fragments contained in the populationof RNA molecules to be sequence, prior to the preparation of thelibrary. By blocking the miR-10b-5p fragments before the library isgenerated, the sequencing capacity for the less abundant small RNAspecies is increased and the efficiency of the sequencing reactionimproved. Fewer resources are wasted during both the library preparationand sequencing steps since the blocked miR-10b-5p fragments will not actas a substrate during the library preparation and will therefore not beread during the sequencing. Further, by using 5′end and/or 3′endmodified oligonucleotide probes which are themselves blocked,incorporation of the oligonucleotide probes into the sequence libraryand the consequential increase in background noise can be avoided.

In another embodiment, disclosed is a method of performing nextgeneration sequencing of small RNA from a sample, comprising:

-   -   adding miR-10b-5p specific oligonucleotide probes to a sample        containing the population of RNA molecules, wherein each        miR-10b-5p specific oligonucleotide probe comprises a nucleotide        sequence that is the complement to a nucleotide sequence of        miR-10b-5p; and    -   forming a complex between one or more miR-10b-5p fragments and a        miR-10b-5p specific oligonucleotide probe to provide a        miR-10b-5p blocked sample;    -   preparing a library using the miR-10b-5p blocked sample; and    -   sequencing the library.

The miR-10b-5p blocking steps can be performed as described in greaterdetail above. In a further embodiment, the miR-10b-5p specificoligonucleotide probe can be modified at the 5′end, the 3′ end or bothends. The miR-10b-5p specific oligonucleotide probe can be similarlymodified as described above for modified miR-10a-5p specificoligonucleotide probes.

The library preparation steps and sequencing steps can be performed inaccordance with known NGS protocols.

In one embodiment, the disclosed method of performing next generationsequencing may comprise the preparation of a library using a miR-10a-5pdepleted population of small RNA molecules or a miR-10b-5p depletedpopulation of small RNA molecules. In an alternate embodiment, themethod may comprise the preparation of a library using a miR-10a-5p andmiR-10b-5p depleted population of small RNA molecules, wherein the stepsof depleting the miR-10a-5p fragments and the miR-10b-5p fragments arecarried out successively or concurrently.

In another embodiment, the disclosed method of performing nextgeneration sequencing may comprise the preparation of a library using amiR-10a-5p blocked sample or a miR-10b-5p blocked sample. In analternate embodiment, the method may comprise the preparation of alibrary using a miR-10a-5p blocked and a miR-10b-5p blocked sample,wherein the steps of selectively blocking the miR-10a-5p fragments andthe miR-10b-5p fragments are carried out successively or concurrently.

Kits for Improving Global Gene Expression Analysis

Further disclosed, is a kit for improving global gene expressionanalysis for a population of RNA molecules derived from human urine. Thekit can comprise one or more miR-10a-5p specific oligonucleotide probes,wherein each miR-10a-5p specific oligonucleotide probe comprises anucleotide sequence that is the complement to a nucleotide sequence ofmiR-10a-5p and/or one or more miR-10b-5p specific oligonucleotideprobes, wherein each miR-10b-5p specific oligonucleotide probe comprisesa nucleotide sequence that is the complement to a nucleotide sequence ofmiR-10b-5p.

The kit may comprise any of the miR-10a-5p specific oligonucleotideprobes described in greater detail above. The miR-10a-5p specificoligonucleotide probes can be used to block the miR-10a-5p fragments byforming miR-10a-5p:oligonucleotide complexes. In a preferred embodiment,such miR-10a-5p specific oligonucleotide probes include a 5′end and/or3′end modification to prevent ligation of the probes. For example, the5′end or the 3′end can be modified by incorporating a dideoxy nucleotideas described in greater detail above.

Alternatively, the miR-10a-5p specific oligonucleotide probes can beused to form miR-10a-5p:oligonucleotide complexes, which aresubsequently removed from the population of RNA molecules. ThemiR-10a-5p specific oligonucleotide probes may include a modification tofacilitate removal of the miR-10a-5p:oligonucleotide complex from asample using a solid support. For example, the 5′end or the 3′end of theoligonucleotide probe can be modified with biotin for use with avidin orstreptavidin coupled solid supports. Further examples of suitablemodifications for use with solid supports are described in greaterdetail above.

The kit may comprise any of the miR-10b-5p specific oligonucleotideprobes described in greater detail above. The miR-10b-5p specificoligonucleotide probes can be used to block the miR-10b-5p fragments byforming miR-10b-5p:oligonucleotide complexes. In a preferred embodiment,such miR-10b-5p specific oligonucleotide probes include a 5′end and/or3′end modification to prevent ligation of the probes. For example, the5′end or the 3′end can be modified by incorporating a dideoxy nucleotideas described in greater detail above. The same modifications describedabove for miR-10a-5p specific oligonucleotide probes can also beincorporated into miR-10b-5p specific oligonucleotide probes.

The miR-10b-5p specific oligonucleotide probes can also be used to formmiR-10b-5p:oligonucleotide complexes, which are subsequently removedfrom the population of RNA molecules. The miR-10b-5p specificoligonucleotide probes may include a modification to facilitate removalof the miR-10b-5p:oligonucleotide complex from a sample using a solidsupport. For example, the 5′end or the 3′end of the miR-10b-5p specificoligonucleotide probe can be modified with biotin for use with avidin orstreptavidin coupled solid supports. Further examples of suitablemodifications for use with solid supports are described in greaterdetail above. The same modifications described above for miR-10a-5pspecific oligonucleotide probes can also be incorporated into miR-10b-5pspecific oligonucleotide probes.

In another embodiment, the miR-10a-5p specific oligonucleotide probesand/or the miR-10b-5p specific oligonucleotide probes can be providedimmobilized on a solid support, such as purification beads, which may bemagnetic or non-magnetic.

The kit may be designed for use in the selective depletion, selectiveblocking, sequencing and library generation methods as described aboveand may comprise further components. Each component may be provided inseparate compartments or vessels. The kit may also be provided withinstructions for using the kit, or with directions for how instructionsmay be obtained.

Although the invention has been described with reference to illustrativeembodiments, it is to be understood that the invention is not limited tothese precise embodiments, and that various changes and modification areto be intended to be encompassed in the appended claims.

EXAMPLES

These examples are described for the purposes of illustration and arenot intended to limit the scope of the invention.

Example 1 Preparation of Capture Probe for miR-10a-5p Fragment

The capture probe for the miR-10a-5p fragment was designed by using the“Homo sapiens miRNA 10a” (miRBase accession: MIMAT0000253) as areference sequence for the full length miR-10a-5p and creating acomplement of the 23 nucleotides in the sequence. This was based onprevious observations and sequencing data of small RNA purified fromhuman urine, which showed that the second most over-represented sequencein the small RNA fraction of RNA purified from urine was the 23nucleotides long miR-10a-5p (El-Mogy et al., 2018).

The capture probe was designed to be the complement of the 23 basemiR-10a-5p fragment having the sequence

(SEQ ID NO: 1) 5′- UACCCUGUAGAUCCGAAUUUGUG -3′.

The sequence of the oligonucleotide capture probe is:

(SEQ ID NO: 2) 5′- CACAAATTCGGATCTACAGGGTA -3′.

In order to facilitate the removal of the miR-10a-5p:captureoligonucleotide complexes, biotin was covalently attached to the 5′ endof the capture oligonucleotide.

Example 2 Depletion of the miR-10a-5p Fragment from Human Urine

A 30 mL urine sample was collected into a 50 cc Falcon tube (BDDiagnostics, Mississauga, Canada) from 6 healthy donors (3 males and 3females). Total RNA was then purified from the 30 mL of the human urineusing Norgen's Urine Cell-Free Circulating RNA Purification Maxi Kit(Cat# 57100, Norgen Biotek Corp., Thorold, Canada) according to theprovided protocol.

Next, the miR-10a-5p fragment was depleted from the total RNA sampleusing the probe described in Example 1. Briefly, Streptavidin MagneticBeads were prepared by aliquoting 125 μL (500 μg) of StreptavidinMagnetic Beads (New England Biolabs, Whitby, Canada) into a cleanRNase-free microcentrifuge tube, and 100 μL of buffer [0.5 M NaCl, 20 mMTris-HCl (pH 7.5), 1 mM EDTA] was added to the beads and they were thenvortexed to suspend. A magnet was then applied to the side of tube forapproximately 30 seconds, and the supernatant was removed and discarded.Next, 1.0 A₂₆₀ unit of the biotin-(miR-10a-5p fragment capture probe)was dissolved in in 500 μL of buffer [0.5 M NaCl, 20 mM Tris-HCl (pH7.5), 1 mM EDTA] to a final concentration 8 pmol/μL. Next, 25 μL of thebiotin-(miR-10a-5p fragment capture probe) solution was added to theprepared magnetic beads and vortexed to suspend the beads. This was thenincubated at room temperature for 5 minutes with occasional agitation byhand, then a magnet was applied, and the supernatant was again removedand discarded. The beads were washed by adding 100 μL of buffer [0.5 MNaCl, 20 mM Tris-HCl (pH 7.5), 1 mM EDTA], vortexing to suspend, andthen applying a magnet and discarding the supernatant. The beads werethen washed a second time in the same manner.

Next, 25 μL of the total RNA purified from urine was mixed with 25 μL ofbuffer [1 M NaCl, 40 mM Tris-HCl (pH 7.5), 2 mM EDTA] and heated at 65°C. for 5 minutes then quickly chilled at 4° C. for 3 minutes. The totalRNA sample was then added to the previously prepared magnetic beads. Themixture was vortexed to suspend the particles, then incubated at roomtemperature for 10 minutes with occasional agitation by hand. A magnetwas then applied and the supernatant (containing the depleted RNA) wascollected. Next, 100 μL of the buffer was again added to the beads,followed by vortexing to suspend the beads. Again, a magnet was appliedand the supernatant (containing the depleted RNA) was collected. Thisprocess was then repeated, for a total of 3 collections of the depletedRNA. Finally, 100 μL of a cold low salt buffer [0.15 M NaCl, 20 mMTris-HCl (pH 7.5), 1 mM EDTA] was added to beads, and vortexed tosuspend. Again, a magnet was applied, and the supernatant was removedand collected. All the recovered supernatants were then pooled.

The miR-10a-5p fragment-depleted RNA can be assayed or further processed(e.g. preparation of a sequencing library) immediately or it can bepurified prior to the assay. Multiple purification and concentrationmethods are possible, including through the use of silicon carbidecolumns, silica columns, gel electrophoresis or ethanol precipitation.

Example 3 Improved Ratio of Useful Data Obtained During Small RNA NextGeneration Sequencing of Human Urine by Selectively Depleting the HighlyAbundant miR-10a-5p Fragments

Two 30 mL urine samples were collected into 50 cc Falcon tubes (BDDiagnostics, Mississauga, Canada) from 6 healthy donors (3 males and 3females)—a total of 12 tubes were collected. Total RNA was then purifiedfrom the 30 mL of human urine using Norgen's Urine Total RNAPurification Maxi Kit (Slurry Format) (Cat# 29600, Norgen Biotek Corp.,Thorold, Canada) according to the provided protocol. Next, themiR-10a-5p fragment was depleted from one of the total RNA samples fromeach donor using the probe described in Example 1 and the methodoutlined in Example 2. The other sample from each donor was not depletedin order to be used as a control.

Samples of miR-10a-5p fragment-depleted RNA were then concentrated usingNorgen's Urine Total RNA Purification Maxi Kit (Slurry Format) (Cat#29600, Norgen Biotek Corp., Thorold, Canada) with a slight modificationto the first two steps in the provided protocol: 1) The miR-10a-5pfragment-depleted RNA was mixed with an equal volume of Lysis Buffer A;and 2) the resulting mixture was then mixed with an equal volume of96-100% ethanol (for example, a 350 μL RNA sample depleted of miR-10a-5pwas first mixed with 350 μL of Lysis Buffer A and then mixed with 700 μLof 96-100% ethanol). Subsequently, the provided protocol was followed asspecified in the kit insert of Norgen's Urine Total RNA PurificationMaxi Kit (Slurry Format) (Cat# 29600, Norgen Biotek Corp., Thorold,Canada), starting with Step 3.

The concentrated miR-10a-5p fragment-depleted RNA from each donor wasthen used for small RNA library preparation for downstream NGS analysis.Briefly, using the NEBNext® Multiplex Small RNA Library Prep Set forIllumina® (New England Biolabs, Whitby, Canada), the RNA was firstligated to the 3′ adapter, followed by RT primer hybridization and 3′adapter blocking. Next, the 5′ adapter was ligated to the 5′ end of theRNA, which was then reverse transcribed into cDNA. This was followed bya limited (15) cycle PCR amplification to enrich the cDNA and also toattach the indexing (barcode) sequences. The indexed libraries were thenresolved on a 6% TBE gel and the fragments of interest excised from thegel, crushed and left over-night in 200 μL of water to release DNA. Thecrushed gel pieces were filtered out and the DNA was concentrated usingNorgen's RNA Clean-Up and Concentration Micro-Elute Kit (Cat# 61000,Norgen, Thorold, Canada) according to the provided protocol. Alllibraries were quantified and assessed for library size by the AgilentBioanalyzer using the Agilent High Sensitivity DNA Kit (AgilentTechnologies, Santa Clara, United States). As a control, the urine RNAisolated from each individual that was not depleted of the miR-10a-5pfragment was also used for small RNA library preparation.

Next, all of the small RNA libraries were sequenced on the IlluminaNextSeq® (Illumina Inc., San Diego, United States) instrument accordingto the instructions provided by the manufacturer (Preparing Librariesfor Sequencing on the NextSeq® and the NextSeq® System User Guide). Theresulting NGS sequencing data was then analyzed in a number of differentways to verify that the ratio of useful data obtained was improved inthe small RNA libraries prepared from urine that was depleted ofmiR-10a-5p compared to the control small RNA libraries prepared fromnon-depleted urine.

The average number of reads per million mapped to miRNA was graphed forthe control (non-depleted) and miR-10a-5p-depleted samples (FIG. 1).Depletion of miR-10a-5p increased the NGS run capacity towards othermiRNA sequences and therefore enhanced run outcomes. The number of readsthat represents miRNA sequences in an NGS library has increased afterthe depletion of miR-10a-5p. FIG. 2 illustrates the average number ofreads graphed according to insert size incorporated into the library.When performing NGS of small RNA libraries from human urine, the mainRNA of interest for analysis is miRNA, which are approximately 20 nt insize. The abundant miR-10a-5p fragment is 23 nt in size. Therefore, thedepletion of the miR-10a-5p fragment can also be verified by determiningthe % of reads for each insert size. These results demonstrate that themethod of the present invention improves the NGS run capacity.

Example 4 Preparation of Capture Probe for miR-10b-5p Fragment

The capture probe for the miR-10b-5p fragment was designed by using the“Homo sapiens miRNA 10b” (miRBase accession: MIMAT0000254) as areference sequence for the full length miR-10b-5p and creating acomplement of the 23 nucleotides in the sequence. This was based onprevious observations and sequencing data of small RNA purified formurine, which showed that the most over-represented sequence in the smallRNA fraction of RNA purified from urine was the 23 nucleotide longmiR-10b-5p.

The capture probe was designed to be the complement of the 23 basemiR-10b-5p fragment having the sequence

(SEQ ID NO: 3) 5′- UACCCUGUAGAACCGAAUUUGUG-3′.

The sequence of the oligonucleotide capture probe is:

(SEQ ID NO: 4) 5′- CACAAATTCGGTTCTACAGGGTA -3′.

In order to facilitate the removal of the miR-10b-5p:captureoligonucleotide complexes, biotin was covalently attached to the 5′ endof the capture oligonucleotide.

Example 5 Depletion of the miR-10b-5p Fragment from Human Urine

A 30 mL urine sample was collected into a 50 cc Falcon tube (BDDiagnostics, Mississauga, Canada) from 6 healthy donors (3 males and 3females). Total RNA was then purified from the 30 mL of the human urineusing Norgen's Urine Cell-Free Circulating RNA Purification Maxi Kit(Cat# 57100, Norgen Biotek Corp., Thorold, Canada) according to theprovided protocol.

Next, the miR-10b-5p fragment was depleted from the total RNA sampleusing the probe described in Example 4. Briefly, Streptavidin MagneticBeads were prepared by aliquoting 125 μL (500 μg) of StreptavidinMagnetic Beads (New England Biolabs, Whitby, Canada) into a cleanRNase-free microcentrifuge tube, and 100 μL of buffer [0.5 M NaCl, 20 mMTris-HCl (pH 7.5), 1 mM EDTA] was added to the beads and they were thenvortexed to suspend. A magnet was then applied to the side of tube forapproximately 30 seconds, and the supernatant was removed and discarded.Next, 1.0 A₂₆₀ unit of the biotin-( miR-10b-5p fragment capture probe)was dissolved in in 500 μL of buffer [0.5 M NaCl, 20 mM Tris-HCl (pH7.5), 1 mM EDTA] to a final concentration 8 pmol/μL. Next, 25 μL of thebiotin-(miR-10b-5p fragment capture probe) solution was added to theprepared magnetic beads and vortexed to suspend beads. This was thenincubated at room temperature for 5 minutes with occasional agitation byhand, then a magnet was applied, and the supernatant was again removedand discarded. The beads were washed by adding 100 μL of buffer [0.5 MNaCl, 20 mM Tris-HCl (pH 7.5), 1 mM EDTA], vortexing to suspend, andthen applying a magnet and discarding the supernatant. The beads werethen washed a second time in the same manner.

Next, 25 μL of the total RNA purified from urine was mixed with 25 μL ofbuffer [1 M NaCl, 40 mM Tris-HCl (pH 7.5), 2 mM EDTA] and heated at 65°C. for 5 minutes then quickly chilled at 4° C. for 3 minutes. The totalRNA sample was then added to the previously prepared magnetic beads. Themixture was vortexed to suspend the particles, then incubated at roomtemperature for 10 minutes with occasional agitation by hand. A magnetwas then applied and the supernatant (containing the depleted RNA) wascollected. Next, 100 μL of the buffer was again added to the beads,followed by vortexing to suspend the beads. Again, a magnet was appliedand the supernatant (containing the depleted RNA) was collected. Thisprocess was then repeated, for a total of 3 collections of the depletedRNA. Finally, 100 μL of a cold low salt buffer [0.15 M NaCl, 20 mMTris-HCl (pH 7.5), 1 mM EDTA] was added to beads, and vortexed tosuspend. Again, a magnet was applied, and the supernatant was removedand collected. All of the recovered supernatants were then pooled.

The miR-10b-5p fragment-depleted RNA can be assayed or further processed(e.g. preparation of a sequencing library) immediately or it can bepurified prior to the assay. Multiple purification and concentrationmethods are possible, including through the use of silicon carbidecolumns, silica columns, gel electrophoresis or ethanol precipitation.

Example 6 Improved Ratio of Useful Data Obtained During Small RNA NextGeneration Sequencing of Human Urine by Selectively Depleting the HighlyAbundant miR-10b-5p Fragments

Two 30 mL urine samples were collected into 50 cc Falcon tubes (BDDiagnostics, Mississauga, Canada) from 6 healthy donors (3 males and 3females)—a total of 12 tubes were collected. Total RNA was then purifiedfrom the 30 mL of the human urine using Norgen's Urine Total RNAPurification Maxi Kit (Slurry Format) (Cat# 29600, Norgen Biotek Corp.,Thorold, Canada) according to the provided protocol. Next, themiR-10b-5p fragment was depleted from one of the total RNA samples fromeach donor using the probe described in Example 4 and the methodoutlined in Example 5. The other sample from each donor was not depletedin order to be used as a control.

Samples of miR-10b-5p fragment-depleted RNA were then concentrated usingNorgen's Urine Total RNA Purification Maxi Kit (Slurry Format) (Cat#29600, Norgen Biotek Corp., Thorold, Canada) with a slight modificationto the first two steps in the provided protocol: 1) The miR-10a-5pfragment-depleted RNA was mixed with an equal volume of Lysis Buffer A;and 2) the resulting mixture was then mixed with an equal volume of96-100% ethanol (for example, a 350 μL RNA sample depleted of miR-10a-5pwas first mixed with 350 μL of Lysis Buffer A and then mixed with 700 μLof 96-100% ethanol). Subsequently, the provided protocol was followed asspecified in the kit insert of Norgen's Urine Total RNA PurificationMaxi Kit (Slurry Format) (Cat# 29600, Norgen Biotek Corp., Thorold,Canada), starting with Step 3.

The concentrated miR-10b-5p fragment-depleted RNA from each donor wasthen used for small RNA library preparation for downstream NGS analysis.Briefly, using the NEBNext® Multiplex Small RNA Library Prep Set forIllumina® (New England Biolabs, Whitby, Canada), the RNA was firstligated to the 3′ adapter, followed by RT primer hybridization and 3′adapter blocking. Next, the 5′ adapter was ligated to the 5′ end of theRNA, which was then reverse transcribed into cDNA. This was followed bya limited (15) cycle PCR amplification to enrich the cDNA and also toattach the indexing (barcode) sequences. The indexed libraries were thenresolved on a 6% TBE gel and the fragments of interest excised from thegel, crushed and left over-night in 200 μL of water to release DNA. Thecrushed gel pieces were filtered out and the DNA in the filtrateconcentrated using Norgen's RNA Clean-Up and Concentration Micro-EluteKit (Cat# 61000, Norgen, Thorold, Canada) according to the providedprotocol. All libraries were quantified and assessed for library size bythe Agilent Bioanalyzer using the Agilent High Sensitivity DNA Kit(Agilent Technologies, Santa Clara, United States). As a control, theurine RNA isolated from each individual that was not depleted of themiR-10b-5p fragment was also used for small RNA library preparation.

Next, all of the small RNA libraries were sequenced on the IlluminaNextSeq® (Illumina Inc., San Diego, United States) instrument accordingto the instructions provided by the manufacturer (Preparing Librariesfor Sequencing on the NextSeq® and the NextSeq® System User Guide). Theresulting NGS sequencing data was then analyzed in a number of differentways to verify that the ratio of useful data obtained was improved inthe small RNA libraries prepared from urine that was depleted of the 5′fragment of miR-10b-5p compared to the control small RNA librariesprepared from non-depleted urine.

The average number of reads per million mapped to miRNA was graphed forthe control (non-depleted) and miR-10b-5p-depleted samples (FIG. 3).Depletion of miR-10b-5p increased the NGS run capacity towards othermiRNA sequences and therefore enhanced run outcomes. The number of readsthat represents miRNA sequences in an NGS library has increased afterthe depletion of miR-10b-5p. FIG. 4 illustrates the average number ofreads graphed according to insert size incorporated into the library.When performing NGS of small RNA libraries from urine, the main RNA ofinterest for analysis is miRNA, which are approximately 20 nt in size.The abundant miR-10b-5p fragment is 23 nt in size. Therefore, thedepletion of the miR-10b-5p fragment can also be verified by determiningthe % of reads for each insert size. FIG. 5 is a graph depicting theaverage number of miRNA detected in NGS runs from libraries created fromboth control (non-depleted) urine RNA, as well as the miR-10b-5pfragment-depleted urine RNA. Three minimum counts cut-offs were used toconsider a miRNA detectable: 2 counts, 5 counts and 10 counts. Thenumber of detected miRNAs was increased upon miR-10b-5p depletion by 45,48 and 55 miRNAs at the three detection cut-off reads used (2, 5 and 10counts, respectively), with 28-37% increase in numbers of detectedmiRNAs. Fragment-depletion resulted in a greater sensitivity of miRNAdetection because of increased sequencing depth. These resultsdemonstrate that the method of the present invention improves thesignal-to-noise ratio and allows for more low-abundance miRNAs to bedetected during NGS applications.

Example 7 Depletion of the miR-10b-5p and miR-10b-5p Fragments fromHuman Urine

A 30 mL urine sample was collected into a 50 cc Falcon tube (BDDiagnostics, Mississauga, Canada) from 6 healthy donors (3 males and 3females). Total RNA was then purified from the 30 mL of the human urineusing Norgen's Urine Cell-Free Circulating RNA Purification Maxi Kit(Cat# 57100, Norgen Biotek Corp., Thorold, Canada) according to theprovided protocol.

Next, both the miR10a-5p and miR-10b-5p fragment were depleted from thetotal RNA sample using the probes described in Examples 1 and 4.Briefly, Streptavidin Magnetic Beads were prepared by aliquoting 125 μL(500 μg) of Streptavidin Magnetic Beads (New England Biolabs, Whitby,Canada) into a clean RNase-free microcentrifuge tube, and 100 μL ofbuffer [0.5 M NaCl, 20 mM Tris-HCl (pH 7.5), 1 mM EDTA] was added to thebeads and they were then vortexed to suspend. A magnet was then appliedto the side of tube for approximately 30 seconds, and the supernatantwas removed and discarded. Next, 1.0 A₂₆₀ unit of the biotin-(miR10a-5pand miR-10b-5p fragment capture probe) was dissolved in in 500 μL ofbuffer [0.5 M NaCl, 20 mM Tris-HCl (pH 7.5), 1 mM EDTA] to a finalconcentration 8 pmol/μL. Next, 25 μL of the biotin-(miR10a-5p andmiR-10b-5p fragment capture probe) solution was added to the preparedmagnetic beads and vortexed to suspend beads. This was then incubated atroom temperature for 5 minutes with occasional agitation by hand, then amagnet was applied, and the supernatant was again removed and discarded.The beads were washed by adding 100 μL of buffer [0.5 M NaCl, 20 mMTris-HCl (pH 7.5), 1 mM EDTA], vortexing to suspend, and then applying amagnet and discarding the supernatant. The beads were then washed asecond time in the same manner.

Next, 25 μL of the total RNA purified from urine was mixed with 25 μL ofbuffer [1 M NaCl, 40 mM Tris-HCl (pH 7.5), 2 mM EDTA] and heated at 65°C. for 5 minutes then quickly chilled at 4° C. for 3 minutes. The totalRNA sample was then added to the previously prepared magnetic beads. Themixture was vortexed to suspend the particles, then incubated at roomtemperature for 10 minutes with occasional agitation by hand. A magnetwas then applied and the supernatant (containing the depleted RNA) wascollected. Next, 100 μL of the buffer was again added to the beads,followed by vortexing to suspend the beads. Again, a magnet was appliedand the supernatant (containing the depleted RNA) was collected. Thisprocess was then repeated, for a total of 3 collections of the depletedRNA. Finally, 100 μL of a cold low salt buffer [0.15 M NaCl, 20 mMTris-HCl (pH 7.5), 1 mM EDTA] was added to beads, and vortexed tosuspend. Again, a magnet was applied, and the supernatant was removedand collected. All the recovered supernatants were then pooled.

The miR10a-5p and miR-10b-5p fragment-depleted RNA can be assayed orfurther processed (e.g. preparation of a sequencing library) immediatelyor it can be purified prior to the assay. Multiple purification andconcentration methods are possible, including through the use of siliconcarbide columns, silica columns, gel electrophoresis or ethanolprecipitation.

Example 8 Improved Ratio of Useful Data Obtained During Small RNA NextGeneration Sequencing of Human Urine by Selectively Depleting the HighlyAbundant miR10a-5p and miR-10b-5p Fragments

Two 30 mL urine samples were collected into 50 cc Falcon tubes (BDDiagnostics, Mississauga, Canada) from 6 healthy donors (3 males and 3females), a total of 12 tubes were collected. Total RNA was thenpurified from the 30 mL of the human urine using Norgen's Urine TotalRNA Purification Maxi Kit (Slurry Format) (Cat# 29600, Norgen BiotekCorp., Thorold, Canada) according to the provided protocol. Next, themiR10a-5p and miR-10b-5p fragments were depleted from one of the totalRNA samples from each donor using the probes described in Example 1 and4, and the method outlined in Example 7. The other sample from eachdonor was not depleted in order to be used as a control.

Samples of miR-10a-5p and miR-10b-5p fragments-depleted RNA were thenconcentrated using Norgen's Urine Total RNA Purification Maxi Kit(Slurry Format) (Cat# 29600, Norgen Biotek Corp., Thorold, Canada) witha slight modification to the first two steps in the providedprotocol: 1) The miR-10a-5p and miR-10b-5p fragments-depleted RNA wasmixed with an equal volume of Lysis Buffer A; and 2) the resultingmixture was then mixed with an equal volume of 96-100% ethanol (forexample, a 350 μL RNA sample depleted of miR-10a-5p and miR-10b-5p wasfirst mixed with 350 μL of Lysis Buffer A and then mixed with 700 μL of96-100% ethanol). Subsequently, the provided protocol was followed asspecified in the kit insert of Norgen's Urine Total RNA PurificationMaxi Kit (Slurry Format) (Cat# 29600, Norgen Biotek Corp., Thorold,Canada), starting with Step 3.

The concentrated miR10a-5p and miR-10b-5p fragment-depleted RNA fromeach donor was then used for small RNA library preparation fordownstream NGS analysis. Briefly, using the NEBNext® Multiplex Small RNALibrary Prep Set for Illumina® (New England Biolabs, Whitby, Canada),the RNA was first ligated to the 3′ adapter, followed by RT primerhybridization and 3′ adapter blocking. Next, the 5′ adapter was ligatedto the 5′ end of the RNA, which was then reverse transcribed into cDNA.This was followed by a limited (15) cycle PCR amplification to enrichthe cDNA and also to attach the indexing (barcode) sequences. Theindexed libraries were then resolved on a 6% TBE gel and the fragmentsof interest excised from the gel, crushed and left over-night in 200 μLof water to release DNA. The crushed gel pieces were filtered out andthe DNA in the filtrate concentrated using Norgen's RNA Clean-Up andConcentration Micro-Elute Kit (Cat# 61000, Norgen, Thorold, Canada)according to the provided protocol. All libraries were quantified andassessed for library size by the Agilent Bioanalyzer using the AgilentHigh Sensitivity DNA Kit (Agilent Technologies, Santa Clara, UnitedStates). As a control, the urine RNA isolated from each individual thatwas not depleted of the miR10a-5p and miR-10b-5p fragment was also usedfor small RNA library preparation.

Next, all of the small RNA libraries were sequenced on the IlluminaNextSeq® (Illumina Inc., San Diego, United States) instrument accordingto the instructions provided by the manufacturer (Preparing Librariesfor Sequencing on the NextSeq® and the NextSeq® System User Guide). Theresulting NGS sequencing data was then analyzed in a number of differentways to verify that the ratio of useful data obtained was improved inthe small RNA libraries prepared from urine that was depleted of themiR10a-5p and miR-10b-5p compared to the control small RNA librariesprepared from non-depleted urine.

FIG. 6 illustrates the average number of reads graphed according toinsert size incorporated into the library. When performing NGS of smallRNA libraries from human urine, the main RNA of interest for analysis ismiRNA, which are approximately 20 nt in size. The abundant miR10a-5p andmiR-10b-5p fragments are 23 nt in size. Therefore, the depletion of themiR10a-5p and miR-10b-5p fragments can also be verified by determiningthe % of reads for each insert size. FIG. 7 is a graph depicting theaverage number of miRNA detected in NGS runs from libraries created fromboth control (non-depleted) urine RNA, as well as the miR10a-5p andmiR-10b-5p fragment-depleted urine RNA. Three minimum counts cut-offswere used to consider a miRNA detectable: 2 counts, 5 counts and 10counts. The number of detected miRNAs was increased upon miR10a-5p andmiR-10b-5p depletion by 99, 64 and 57 miRNAs at the three detectioncut-off reads used (2, 5 and 10 counts, respectively), with 41-51%increase in numbers of detected miRNAs. Fragment-depletion resulted in agreater sensitivity of miRNA detection because of increased sequencingdepth. These results demonstrate that the method of the presentinvention improves the signal-to-noise ratio and allows for morelow-abundance miRNAs to be detected during NGS applications.

REFERENCES

Abdelmaksoud-Dammak R, Chamtouri N, Triki M, Saadallah-Kallel A, AyadiW, Charfi S, et al. Overexpression of miR-10b in colorectal cancerpatients: Correlation with TWIST-1 and E-cadherin expression. TumorBiol. 2017 39(3):1010428317695916.

Arai T, Okato A, Kojima S, Idichi T, Koshizuka K, Kurozumi A, et al.Regulation of spindle and kinetochore-associated protein 1 by antitumormiR-10a-5p in renal cell carcinoma. Cancer Science 2017; 108(10):2088-2101.

El-mogy M, Lam B, Haj-ahmad T A, Mcgowan S, Yu D, Nosal L, et al.Diversity and signature of small RNA in different bodily fluids usingnext generation sequencing. BMC Genomics [Internet]. BMC Genomics;2018;1-24. Available from:https://bmcgenomics.biomedcentral.com/articles/10.1186/s12864-018-4785-8

Ma Z, Chen Y, Min L, Li L, Huang H, Li J, et al. Augmented miR-10bexpression associated with depressed expression of its target gene KLF4involved in gastric carcinoma. Int. J. Clin. Exp. Pathol. 2015;8:5071-9.

Veerla S, Lindgren D, Kvist A, Frigyesi A, Staaf J, Persson H, et al.miRNA expression in urothelial carcinomas: Important roles of miR-10a,miR-222, miR-125b, miR-7 and miR-452 for tumor stage and metastasis, andfrequent homozygous losses of miR-31. Int. J. Cancer 2009; 124:2236-2242.

Xiao H, Li H, Yu G, Xiao W, Hu J, Tang K, et al. MicroRNA-10b promotesmigration and invasion through KLF4 and HOXD10 in human bladder cancer.Oncol. Rep. 2014; 31:1832-8.

Zhang L, Sun J, Wang B, Ren J C, Su W, Zhang T. MicroRNA-10b Triggersthe Epithelial-Mesenchymal Transition (EMT) of Laryngeal Carcinoma Hep-2Cells by Directly Targeting the E-cadherin. Appl. Biochem. Biotechnol.2015; 176:33-44.

Zhou K, Spillman M A, Behbakht K, Komatsu J M, Abrahante J E, Hicks D,et al. A method for extracting and characterizing RNA from urine: Fordownstream PCR and RNAseq analysis. Anal. Biochem. 2017; 536:8-15.

1. A method of improving global gene expression analysis for apopulation of RNA molecules derived from human urine, the methodcomprising the step of blocking miR-10a-5p fragments and/or miR-10b-5pfragments in the population of RNA molecules.
 2. The method of claim 1,wherein the step of blocking the miR-10a-5p fragments and/or miR-10b-5pfragments in the population of RNA molecules comprises: addingmiR-10a-5p specific oligonucleotide probes and/or miR-10b-5p specificoligonucleotide probes to a sample containing the population of RNAmolecules, wherein each miR-10a-5p specific oligonucleotide probecomprises a nucleotide sequence that is the complement to a nucleotidesequence of miR-10a-5p and each miR-10b-5p specific oligonucleotideprobe comprises a nucleotide sequence that is the complement anucleotide sequence of miR-10b-5p; and forming a complex between one ormore miR-10a-5p fragments and a miR-10a-5p specific oligonucleotideprobe and/or forming a complex between one or more miR-10b-5p fragmentsand a miR-10b-5p specific oligonucleotide probe to provide a miR-10a-5pand/or miR-10b-5p blocked sample.
 3. The method of claim 2, wherein the5′end, the 3′end or both ends of each miR-10a-5p specificoligonucleotide probes and/or miR-10b-5p specific oligonucleotide probesis modified to prevent ligation and wherein the modification is a 5′biotin modification, a 3′ biotin modification, a 5′ dioxigeninmodification, a 3′ dioxigenin modification, a 5′ dinitrophenolmodification, 5′ dideoxy nucleotide modification, a 3′ dideoxynucleotide modification or a combination thereof.
 4. The method of claim2, wherein the nucleotide sequence of miR-10a-5p has at least 90%identity to the nucleotide sequence of SEQ ID NO: 1 and wherein thenucleotide sequence of miR-10b-5p has at least 90% identity to thenucleotide SEQ ID NO:
 3. 5. The method of claim 2, wherein themiR-10a-5p specific oligonucleotide probe has at least 90% identity tothe nucleotide sequence of SEQ ID NO: 2 and the miR-10b-5p specificoligonucleotide probe has at least 90% identity to the nucleotidesequence of SEQ ID NO:
 4. 6. The method of claim 2, wherein the globalgene expression analysis is next generation sequencing and wherein themethod further comprises the steps of: preparing a library using themiR-10a-5p and/or miR-10b-5p blocked sample; and sequencing the library.